Brain-wide neural dynamics of poststroke recovery induced by optogenetic stimulation

Abstract

Poststroke optogenetic stimulations can promote functional recovery. However, the circuit mechanisms underlying recovery remain unclear. Elucidating key neural circuits involved in recovery will be invaluable for translating neuromodulation strategies after stroke. Here, we used optogenetic functional magnetic resonance imaging to map brain-wide neural circuit dynamics after stroke in mice treated with and without optogenetic excitatory neuronal stimulations in the ipsilesional primary motor cortex (iM1). We identified key sensorimotor circuits affected by stroke. iM1 stimulation treatment restored activation of the ipsilesional corticothalamic and corticocortical circuits, and the extent of activation was correlated with functional recovery. Furthermore, stimulated mice exhibited higher expression of axonal growth-associated protein 43 in the ipsilesional thalamus and showed increased Synaptophysin⁺/channelrhodopsin⁺ presynaptic axonal terminals in the corticothalamic circuit. Selective stimulation of the corticothalamic circuit was sufficient to improve functional recovery. Together, these findings suggest early involvement of corticothalamic circuit as an important mediator of poststroke recovery.

Fig. 1. The experimental timeline and procedures.

(A) Adult C57BL/6J mice first underwent a stereotaxic surgery for virus injection and optical fiber implantation to target iM1 layer V excitatory neurons. Behavioral response to iM1 stimulation was confirmed at 4 weeks after injection. Sensorimotor behavioral testing comprising rotating beam tests were conducted at prestroke, PD4, PD7, PD14, and PD21. Mice underwent transient MCAO on day 0. ofMRI sessions were performed at the following time points to obtain global brain circuit activation maps: prestroke and PD3, PD15, and PD29. Two groups of mice were used: NoStim group (n = 8) and Stim group (n = 9). Stim group received daily stimulations between PD5 and PD14. Mice were euthanized on PD15 and PD30 for Western blots and histology, respectively. (B) Functional MRI scans covering the whole brain (27 coronal slices as shown by small dots) were acquired during 10-Hz iM1 stimulation. The ofMRI stimulation paradigm consisted of two runs, each comprising six blocks of 15-s on and 40-s off periods. (C) OST paradigm: The Stim group received daily iM1 stimulations consisting of three 1-min stimulation periods with 3-min rest periods in between each stimulation. NoStim group went through identical procedures without any stimulations. (D) Diagram illustrates the optical fiber implant location in iM1 where ChR2-eYFP was expressed. (E) Representative images illustrating histological validation of infarct size using immunohistochemistry with neuronal marker MAP2 (left) and inflammatory activation marker CD68 (middle); combined MAP2 and CD68 (right). All mice used in this study exhibited infarcts in both the ipsilesional striatum and S1. Scale bar, 1000 μm.

Fig. 2. iM1 excitatory neuronal stimulation activates a widespread network of cortical and subcortical areas before stroke.

(A) ofMRI activation maps at prestroke baseline. Brain areas were activated by iM1 stimulation at prestroke baseline. Color-coded activation maps indicate z score values (red, increased activation; blue, decreased activation) and are corrected for multiple comparisons using GRF, P < 0.05 (n = 17). Number below each slice indicates distance relative to bregma. (B) Quantitative analysis of activation volume and magnitude in key brain areas activated at prestroke baseline. For each area, left panel shows BOLD signal change time series during the six blocks of iM1 stimulation averaged across Stim mice (n = 9, blue) and NoStim mice (n = 8, red). Green line indicates the on and off stimulation periods. The middle graph shows the BOLD signal change time series averaged across all blocks in each group. The bar plots (right) show the percentage of activated volume in each area in the Stim (blue) and NoStim (red) group. There is no difference in either magnitude of activation (middle) or activation volume (bar plots) between the Stim and NoStim group at prestroke baseline (n.s., not significant; P > 0.4 in all areas). (C) Histological examination confirmed that ChR2-eYFP is expressed in iM1-connected regions, consistent with brain areas that are functionally activated during iM1 stimulation as demonstrated in (A). M2, secondary motor cortex; Str, striatum; cc, corpus callosum; Ac, Anterior cingulate; hpx, hippocampus; TH, thalamus; po, posterior thalamic nucleus; vpm, ventral posteromedial thalamic nucleus; vpl, ventral posterolateral thalamic nucleus; vm, ventromedial thalamic nucleus; cM1, contralesional M1; iTH, ipsilesional thalamusi; iStr, ipsilesional striatum; iS1, ipsilesional primary somatosensory cortex. Scale bar, 1000 μm.

Fig. 3. iM1 excitatory neuronal stimulation promotes functional recovery after stroke.

(A) Images represent T2-weighted MRI on PD3. The color-coded map shows the probability of infarct at different brain areas for mice included in this study (n = 17). Infarct areas were detected in ipsilesional somatosensory, parietal and temporal cortices, striatum, thalamus, and hippocampus. (B) Quantitation of total infarct volume shows no difference between Stim (blue) and NoStim (red) groups (P > 0.9). Data are expressed as means ± SEM with scatter plot of individual mice. (C) Stimulated stroke mice (blue) exhibited significant behavior improvement in their sensorimotor function compared to the NoStim mice (red) on PD14. Speed of traveling is expressed as percent improvement compared to prestroke baseline. n = 8 for NoStim mice, n = 9 for Stim mice. Two-way analysis of variance (ANOVA) with Bonferroni’s post hoc test, *P < 0.05. Data are expressed as means ± SEM.

Fig. 4. iM1 excitatory neuronal stimulation restores activation in the iTH and somatosensory cortex after stroke.

(A) ofMRI activation maps in the NoStim (left) and Stim group (right) at prestroke and PD3, PD15, and PD29. Color-coded activation maps indicate z score values (red, increased activation; blue, decreased activation) and are corrected for multiple comparisons using GRF, P < 0.05 (n = 8 in NoStim group, n = 9 in Stim group). At prestroke and PD3, there is no difference in activation between groups during iM1 stimulation. At PD3, all mice exhibit a depressed response throughout the brain. At PD15 and PD29, the NoStim mice show some recovery of activation locally around the stimulation site at iM1 and in ipsilesional S1, while the stimulated mice show greater recovery of activation around the stimulation site at iM1 and in ipsilesional S1, as well as in remote areas such as the iTH. (B) Quantitative analysis of BOLD signal time series at key brain areas revealed that at PD15, the Stim mice (blue) show significant greater activation in the iTH and S1 areas compared to the NoStim mice (red), both in terms of activation magnitude and volume of activation. Two sample t statistics is used to compare the magnitude of activation (middle) and the activated volume (bar plots) between groups (the related P values are reported on top of each panel, Bonferroni corrected). At PD29, only the ipsilesional S1 activation remains significantly higher in the Stim compared to the NoStim group (magnitude of activation, P < 0.05). Abbreviations are listed in Fig. 2.

Fig. 5. Distinct involvements of the corticothalamic and corticocortical circuits at different phases of recovery following stimulation treatment after stroke.

(A) Color-coded activation maps show brain areas whose activation at PD15 is significantly correlated with the amount of early behavioral improvements in the Stim group. Z score maps are corrected for multiple comparisons using GRF, P < 0.05. (B) At PD15, the BOLD signal change in the iTH, but not in the ipsilesional cortical areas (including M1 and S1), was significantly correlated with early behavioral improvements on a per subject basis exclusively in the Stim group. Abscissas represent the percentage change from PD4 in distance traveled in the rotating beam test at PD14. a.u., arbitrary units. (C) Color-coded activation maps show that activation in the ipsilesional corticocortical circuits on PD29 was significantly correlated with late behavioral improvements in the Stim group. (D) At PD29, the change in the BOLD signal in the ipsilesional cortex, but not in the thalamus, was positively correlated with late behavioral improvements exclusively in the Stim group. Abscissas represent the percentage change from PD4 in distance traveled in the rotating beam test at PD21. **P < 0.01 and *P < 0.05.

Fig. 6. Activation in distinct nuclei of the iTH by iM1 stimulation.

(A) Images represent the spatial overlap between the mask of different thalamic nuclei and the statistical map representing the increased activation from PD3 to PD15 in the stimulated mice. z score maps are corrected for multiple comparisons using GRF, P < 0.05. (B) Quantitative analysis showed that the volume of activation in three ipsilesional thalamic nuclei was significantly recovered in the stimulated mice when compared to the NoStim mice on PD15 (*P < 0.05, Bonferroni corrected). These include PO, VP, and VAL of the thalamus.

Fig. 7. iM1 excitatory neuronal stimulation increases plasticity marker expression in the corticothalamic circuit.

Representative images of ChR2-eYFP expression at the ipsilesional striatal (iM1 and iS1 peri-infarct zone) (A) and thalamic (iTH) (B) coronal slices in the Stim and NoStim groups. Dashed lines indicate selected regions of interest. Scale bar, 500 μm. (C) Graphs show quantification of ChR2 expression in the site of injection in iM1, iS1 peri-infarct zone, and the iTH in the Stim (blue) and NoStim (red) groups. The ordinate represents the average fluorescence intensity in the corresponding region of interest subtracted from the average background intensity. Stimulated mice exhibit significantly higher axonal terminal expression of ChR2 in iTH, when compared to the NoStim group (two-sample t statistics, **P < 0.01), while the ChR2 expression level is comparable between groups at the injection site in iM1 (P > 0.25). Scale bar, 100 μm. (D) Western blot analyses on the iTH and contralesional thalamus (cTH) tissues show that stimulated mice exhibit a significant increase in plasticity marker GAP43 protein levels in iTH (two-sample t statistics, *P < 0.05), but not in the contralesional thalamus. (E) Representative high magnification images of ChR2 (green) and Synaptophysin, a presynaptic marker (red) in the thalamus of NoStim (left) and Stim (right) group. (F) Stim mice showed significant increase in the colocalization of ChR2⁺/Synaptophysin⁺ axonal presynaptic terminals in iTH when compared to NoStim mice, as evaluated by Manders’ overlap coefficient (two-sample t statistics, *P < 0.05). (G) Furthermore, the number of ChR2⁺/Synaptophysin⁺ presynaptic terminals in iTH is significantly higher in the Stim group when compared to the NoStim group (two-sample t statistics, *P < 0.05). n = 8 for NoStim mice, n = 9 for Stim mice.

Fig. 8. Selective stimulation of the corticothalamic circuit is sufficient to improve functional recovery after stroke.

(A) Schematic illustrates selective targeting of the corticothalamic circuit: Virus injection (AAV1-CamKIIa-ChR2-eYFP) in iM1 and optical fiber implanted in the iTH. (B) Image represents the spatial overlap between the color-coded mask of different thalamic nuclei and the location of the implanted optical fiber. (C) Representative images at the M1 level (top) and thalamus level (bottom) show the ChR2-eYFP expression (green) locally at the injection site at iM1 and at its axonal projections around PO nucleus in the iTH. GFAP expression (red) illustrates the border of the implanted fiber in the thalamus. Left and right panels show representative images in the Stim and NoStim mice respectively. Scale bar, 500 μm. (D) Representative T2-weighted MR images of the infarct on PD3 in NoStim (top) and Stim (bottom) mice. (E) Quantitation of total infarct volume shows no difference between Stim (blue) and NoStim (red) groups (n = 7 and 6 in the Stim and NoStim underwent imaging on PD3, two-sample t statistics, P > 0.7). Data are expressed as means ± SEM with scatter plot of individual mice. (F) Stimulated stroke mice (blue) exhibited significant behavior improvement in their sensorimotor function compared to the NoStim mice (red) on PD14. n = 10 per group. Two-way ANOVA with Bonferroni’s post hoc test, *P < 0.05.